A new paradigm for identifying patients and drugs at risk for QT prolongation The drug-induced long QT syndrome (diLQTS) continues to be a problem for clinicians balancing risk and benefits across multiple therapeutic areas, and for pharmaceutical scientists and regulators evaluating new drug candidates. The prevalent view is that block of a specific ion current, the rapid component of the cardiac delayed rectifier (IKr), encoded by KCNH2 (also known as HERG), is the common mechanism predisposing to diLQTS across drug classes and that patients with mutations in ion channel genes are at especially increased risk. We present here a body of clinical, genomic, and cellular studies that call these assumptions into question. For example, one important and unexplained clinical feature of diLQTS is that not all IKr blocking drugs confer the same risk; the risk with antiarrhythmics such as sotalol or dofetilide can be 1- 3%, while the risk with IKr-blocking antibiotics such as moxifloxacin is much lower, <1/20,000. Moreover, some drugs, notably phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitors developed for cancer, prolong QT interval but do not block IKr. We and others have recently shown that prolonged (hours) exposure of cardiomyocytes to high risk drugs such as dofetilide generates striking prolongation of action potentials (the in vitro correlate of the QT interval) and arrhythmogenic afterdepolarizations. Importantly, this effect is IKr- independent, does not occur with low risk drugs, and is rapidly and completely reversed by intracellular perfusion with PIP3, implicating inhibition of PI3K as a critical and heretofore unrecognized mechanism contributing to diLQTS risk across drugs. Further, analyses of large international cohorts (whose collection we have led) show that variants in ion channel genes contribute only modestly to risk. Thus a fundamental issue that this Project will address is the extent to which diLQTS risk is predictable in an individual patient. In Specific Aim 1, we will generate cardiomyocytes from induced pluripotent stem cells obtained from patients with a history of diLQTS and from a large cohort of drug-tolerant controls we have ascertained. We will then compare genomic, electrophysiologic, and transcriptomic profiles at baseline (Specific Aim 2) and after drug challenge (Specific Aim 3; PI3K inhibitors; IKr blockers) in cardiomyocytes from subjects with diLQTS and from drug-tolerant controls. The results of these experiments will not only inform the drug development process but will also lead to new methods to screen individual subjects for diLQTS risk.